RESUMO
The present study was undertaken to determine the underlying mechanism of silibinin-induced cell death in human breast cancer cell lines MCF7 and MDA-MB-231. Silibinin-induced cell death was attenuated by antioxidants, N-acetylcysteine (NAC) and 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, suggesting that the effect of silibinin was dependent on generation of reactive oxygen species (ROS). Western blot analysis showed that silibinin induced downregulation of extracellular signal-regulated kinase (ERK) and Akt. When cells were transiently transfected with constitutively active (ca) mitogen-activated protein kinase (MEK), an upstream kinase of ERK and caAkt, they showed resistance to silibinin-induced cell death. Silibinin decreased the cleavage of Notch-1 mRNA and protein levels. Notch-1-overexpressed cells were resistant to the silibinin-induced cell death. Inhibition of Notch-1 signaling was dependent on ROSgeneration. Overexpression of Notch-1 prevented silibinin-induced inhibition of ERK and Akt phosphorylation. Silibinin-induced cell death was accompanied by increased cleavage of caspase-3 and was prevented by caspase-3 inhibitor in MDA-MB-231 cells but not in MCF7 cells. Silibinin induced translocation of apoptosis-inducing factor (AIF), which was blocked by NAC, and transfection of caMEK and caAkt. Silibinin-induced cell death was prevented by silencing of AIF expression using small interfering AIF RNA in MCF7 cells but not in MDA-MB-231 cells. In conclusion, silibinin induces cell death through an AIF-dependent mechanism in MCF7 cells and a caspase-3-dependent mechanism in MDA-MB-231 cells, and ROS generation and Notch-1 signaling act upstream of the ERK and Akt pathway. These data suggest that silibinin may serve as a potential agent for induction of apoptosis in human breast cancer cells.
Assuntos
Antineoplásicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor Notch1/metabolismo , Silimarina/farmacologia , Transporte Ativo do Núcleo Celular , Antioxidantes/farmacologia , Fator de Indução de Apoptose/metabolismo , Neoplasias da Mama , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Regulação para Baixo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Proteínas Proto-Oncogênicas c-akt/genética , Receptor Notch1/genética , Transdução de Sinais , SilibinaRESUMO
Adenosine A(3) receptor (A3AR) is coupled to G proteins that are involved in a variety of intracellular signaling pathways and physiological functions. 2-Chloro-N(6)-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide (Cl-IB-MECA), an agonist of A3AR, has been reported to induce cell death in various cancer cells. However, the effect of CI-IB-MECA on glioma cell growth is not clear. This study was undertaken to examine the effect of CI-IB-MECA on glioma cell viability and to determine its molecular mechanism. CI-IB-MECA inhibited cell proliferation and induced cell death in a dose- and time-dependent manner. Treatment of CI-IB-MECA resulted in an increase in intracellular Ca(2+) followed by enhanced reactive oxygen species (ROS) generation. EGTA and N-acetylcysteine (NAC) blocked the cell death induced by CI-IB-MECA, suggesting that Ca(2+) and ROS are involved in the Cl-IB-MECA-induced cell death. Western blot analysis showed that CI-IB-MECA induced the down-regulation of extracellular signal-regulated kinases (ERK) and Akt, which was prevented by EGTA, NAC, and the A3AR antagonist MRS1191. Transfection of constitutively active forms of MEK, the upstream kinase of ERK, and Akt prevented the cell death. CI-IB-MECA induced caspase-3 activation and the CI-IB-MECA-induced cell death was blocked by the caspase inhibitors DEVD-CHO and z-VAD-FMK. In addition, expression of XIAP and Survivin were decreased in cells treated with Cl-IB-MECA. Collectively, these findings demonstrate that CI-IB-MECA induce a caspase-dependent cell death through suppression of ERK and Akt mediated by an increase in intracellular Ca(2+) and ROS generation in human glioma cells. These suggest that A3AR agonists may be a potential therapeutic agent for induction of apoptosis in human glioma cells.
Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Adenosina/análogos & derivados , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Adenosina/farmacologia , Sequência de Bases , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Primers do DNA , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The flavonoid, luteolin, has been shown to have anticancer activity in various cancer cells; however, the precise molecular mechanism of its action is not completely understood, and studies were conducted to find out how it induces apoptosis in breast cancer cells. Luteolin induced a reduction of viability in a dose- and time-dependent manner. The pro-apoptotic effect of luteolin was demonstrated by cell cycle measurement and Hoechst 3325 staining. Western blot analysis showed that luteolin activates ERK (extracellular-signal-regulated kinase) and p38. Pharmacological inhibition or knockdown of ERK and p38 protected against luteolin-induced cell death; however, the caspase-3-specific inhibitor had no effect. Immunocytochemical examination indicated that luteolin induced nuclear translocation of AIF (apoptosis-inducing factor), which was mediated by activation of ERK and p38. Transfection of a vector expressing the miRNA (microRNA) of AIF prevented luteolin-induced apoptosis. The data suggest that luteolin induces a caspase-dependent and -independent apoptosis involving AIF nuclear translocation mediated by activation of ERK and p38 in breast cancer cells.
Assuntos
Antineoplásicos/farmacologia , Fator de Indução de Apoptose/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Luteolina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/genética , Neoplasias da Mama , Caspase 3/genética , Inibidores de Caspase , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Técnicas de Silenciamento de Genes , Vetores Genéticos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , MicroRNAs/genética , Transporte Proteico/efeitos dos fármacos , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The data presented here are related to the research article entitled "Strengthening and deformation behavior of as-cast CoCrCu1.5MnNi high-entropy alloy (HEA) with micro-/nanoscale precipitation [1]". Non-equimolar CoCrCu1.5MnNi was cast by the conventional induction melting under a high-purity Ar atmosphere. Scanning electron microscopy equipped with energy dispersive spectroscopy (EDS), and transmission electron microscopy (TEM) were used for micro- and nanostructure characterization. Subsize tensile specimens with two different gage length to width ratio were tested at room and cryogenic temperatures to assess the accuracy of strength and ductility data in the as-cast CoCrCu1.5MnNi HEAs. The mixing enthalpy (ΔHmix) versus lattice elastic energy (ΔHel) criterion was used to predict the stable phases. The data on the effects of microstructural and nanostructural distribution of various phases on mechani-cal properties in the as-cast HEA could be used in designing high entropy alloys with excellent as-cast mechanical performance.
RESUMO
Anticancer activity of proteasome inhibitors has been demonstrated in various cancer cell types. However, mechanisms by which they exert anticancer action were not fully understood. The present study was undertaken to examine the effect of the proteasome inhibitor MG-132 and the underlying mechanism in glioma cells. MG-132 caused alterations in mitochondrial membrane potential and apoptosis-inducing factor (AIF) nuclear translocation. MG-132 induced reduction in ERK and Akt activation. The transient transfection of constitutively active forms of MEK, an upstream of ERK, and Akt blocked the MG-132-induced cell death. Similarly to down-regulation of Akt, expression levels of mTOR were inhibited by MG-132. Addition of rapamycin, an inhibitor of mTOR, caused stimulation of the MG-132-induced cell death. There were no significant changes in levels of XIAP, survivin, and Bax. Overexpression of constitutively active forms of MEK and Akt blocked the MG-132-induced AIF nuclear translocation. These findings indicate that MG-132 induces AIF nuclear translocation through down-regulation of ERK and Akt/mTOR pathways. These data suggest that proteasome inhibitors may serve as potential therapeutic agents for malignant human gliomas.
Assuntos
Fator de Indução de Apoptose/metabolismo , Núcleo Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Leupeptinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Imuno-Histoquímica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/fisiologia , Transporte ProteicoRESUMO
Apoptosis of stem cells may be related to certain degenerative conditions such as progressive tissue damage and an inability to repair. Ceramide induces cell death in various cell types. However, the underlying mechanisms of ceramide-induced cell death in stem cells are not explored. This study was designed to investigate the cell death process caused by cell-permeable ceramide and to determine the underlying mechanisms in mesenchymal stem cells derived from human adipose tissue (hASCs). Ceramide caused a loss of cell viability in a concentration- and time-dependent manner, which was largely attributable to apoptosis. Ceramide induced generation of reactive oxygen species (ROS) and disruption of the mitochondrial membrane potential. The ROS generation caused by ceramide was prevented by the antioxidant N-acetylcysteine (NAC). Although ceramide induced release of cytochrome c from mitochondria and activation of caspase-3, the ceramide-induced cell death was partially prevented by caspase inhibitors. Addition of ceramide caused apoptosis-inducing factor (AIF) nuclear translocation, which was prevented by antioxidant. Taken together, these data suggest that ceramide induces cell death through both caspase-dependent and caspase-independent mechanisms mediated by ROS generation in hASCs.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Ceramidas/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Tecido Adiposo/enzimologia , Tecido Adiposo/patologia , Fator de Indução de Apoptose/metabolismo , Técnicas de Cultura de Células , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Ceramidas/fisiologia , Citocromos c/metabolismo , Citometria de Fluxo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The purpose of this study was to establish a prediction rule for severe illness in adult patients hospitalized with pandemic influenza A (H1N1) 2009. At the time of initial presentation, the baseline characteristics of those with severe illness (i.e., admission to intensive care unit, mechanical ventilation, or death) were compared to those of patients with non-severe illnesses. A total of 709 adults hospitalized with pandemic influenza A (H1N1) 2009 were included: 75 severe and 634 non-severe cases. The multivariate analysis demonstrated that altered mental status, hypoxia (PaO(2)/FiO(2) ≤ 250), bilateral lung infiltration, and old age (≥ 65 yr) were independent risk factors for severe cases (all P < 0.001). The area under the ROC curve (0.834 [95% CI, 0.778-0.890]) of the number of risk factors were not significantly different with that of APACHE II score (0.840 [95% CI, 0.790-0.891]) (P = 0.496). The presence of ≥ 2 risk factors had a higher sensitivity, specificity, positive predictive value and negative predictive value than an APACHE II score of ≥ 13. As a prediction rule, the presence of ≥ 2 these risk factors is a powerful and easy-to-use predictor of the severity in adult patients hospitalized with pandemic influenza A (H1N1) 2009.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , APACHE , Adulto , Idoso , Antivirais/uso terapêutico , Feminino , Hospitalização , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/mortalidade , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Pandemias , Valor Preditivo dos Testes , Curva ROC , Respiração Artificial , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
The present study was undertaken to examine the effect of Fructus ligustri lucidi (FLL) extracts on glioma cell growth and to determine the underlying mechanism by which FLL extracts exert anticancer properties in human U87MG glioma cells. The FLL extracts resulted in cell death in a dose- and time-dependent manner. Western blot analysis showed that treatment with FLL extracts caused down-regulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Overexpression of Akt prevented the cell death induced by the FLL extracts. The FLL extracts caused a decrease in the expression of mammalian target of rapamycin (mTOR) and the FLL extract-induced cell death was increased by the mTOR inhibitor rapamycin. The FLL extracts decreased the expression of survivin. Oral administration of FLL extracts in subcutaneous U87MG xenograft models reduced the glioma tumor volume. These findings indicate that the FLL extracts resulted in glioma cell death through regulation of the Akt/mTOR/survivin pathway in vitro and inhibited glioma tumor growth in vivo. These data suggest that the FLL extracts may serve as a potential therapeutic agent for malignant human gliomas.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Glioma/tratamento farmacológico , Ligustrum/química , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Survivina , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mulberry has been reported to contain wide range of polyphenols and have chemopreventive activity. However, little has been known regarding the effect of mulberry fruit extracts on cell viability in vitro in human glioma cells and the anticancer efficacy in vivo. This study was undertaken to examine the effect of mulberry fruit (Moris fructus; MF) extracts on cell viability in vitro and anticancer efficacy in vivo. Cell viability and cell death were estimated by MTT assay and trypan blue exclusion assay, respectively. Reactive oxygen species (ROS) generation was measured using the fluorescence probe DCFH-DA. The mitochondrial transmembrane potential was measured with DiOC(6)(3). Bax expression and cytochrome c release were measured by Western blot analysis. Caspase activity was estimated using colorimetric kit. Cell migration was estimated using the scratched wound model. In vivo anticancer efficacy of MF extracts was evaluated using a subcutaneously injected mouse tumor model. Changes in proliferation and apoptosis were estimated by immunohistochemistric analysis. MF extracts resulted in apoptotic cell death in a dose- and time-dependent manner. MF extracts increased ROS generation, and the MF extract-induced cell death was also prevented by antioxidants, suggesting that ROS generation plays a critical role in the MF extract-induced cell death. Western blot analysis showed that treatment of MF extracts caused an increase in Bax expression, which was inhibited by the antioxidant N-acetylcysteine (NAC). MF extracts induced depolarization of mitochondrial membrane potential, and its effect was inhibited by the antioxidants NAC and catalase. MF extracts induced cytochrome c release, which was inhibited by NAC. Caspase activity was stimulated by MF extracts, and caspase inhibitors prevented the MF extract-induced cell death. Treatment of MF extracts inhibited cell migration. Oral MF extracts administration in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in PCNA-positive cells, an increase in TUNEL-positive cells, and caspase activation. From these data, we concluded that MF extracts reduce glioma tumor growth through inhibition of cell proliferation resulting from induction of apoptosis. These findings suggest that MF extracts result in human glioma cell death in vitro through ROS-dependent mitochondrial pathway and glioma tumor growth in vivo via reduction of tumor cell proliferation and induction of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Glioma/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Morus , Fitoterapia , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Caspases/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Glioma/patologia , Humanos , Mitocôndrias/fisiologia , Proteína X Associada a bcl-2/fisiologiaRESUMO
Rosiglitazone has been reported to exert the protective effect against acute renal failure in animal models. However, the underlying mechanisms by which it protects the damaged kidney cells are poorly understood. The present study was therefore undertaken to examine the effect of rosiglitazone on cell proliferation and to determine its molecular mechanism in opossum kidney (OK) cells, an established renal proximal tubular cell line. Rosiglitazone treatment inhibited cell proliferation in a dose- and time-dependent manner, and such effects were not associated with induction of cell death. The anti-proliferative effect of rosiglitazone was accompanied by the cell cycle arrest at the G1 phase. Western blot analysis data showed that rosiglitazone caused down-regulation of extracellular signal-regulated kinase (ERK) and Akt pathway. Transfection of constitutively active forms of MEK (an upstream kinase of ERK) and Akt prevented the proliferation inhibition induced by rosiglitazone. Rosiglitazone facilitated the recovery of cells after cisplatin-mediated injury. Taken together, these data suggest that rosiglitazone induces inhibition of cell proliferation through ERK and Akt-dependent cell cycle arrest at the G1 phase. The cell cycle arrest may play a protective role in kidney cells by preventing injured cells from progressing in the cell cycle.
Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Tiazolidinedionas/farmacologia , Células Cultivadas , Humanos , RosiglitazonaRESUMO
The present study was undertaken to determine the molecular mechanism by which kaempferol induces cell death in human glioma cells. Kaempferol resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which were largely attributed to cell death. Kaempferol caused an increase in reactive oxygen species (ROS) generation and the kaempferol-induced cell death was prevented by antioxidants, suggesting that ROS generation is involved in kaempferol-induced cell death. Kaempferol caused depolarization of mitochondrial membrane potential. Western blot analysis showed that kaempferol treatment caused a rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. The ERK inhibitor U0126 and the Akt inhibitor LY984002 increased the kaempferol-induced cell death and overexpression of MEK, the upstream kinase of ERK, and Akt prevented the cell death. The expression of anti-apoptotic proteins XIAP and survivin was down-regulated by kaempferol and its effect was prevented by overexpression of MEK and Akt. Kaempferol induced activation of caspase-3 and kaempferol-induced cell death was prevented by caspase inhibitors. Taken together, these findings suggest that kaempferol results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of XIAP and survivin regulating by ERK and Akt.
Assuntos
Antineoplásicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Quempferóis/farmacologia , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Neoplasias Encefálicas , Caspase 3/metabolismo , Inibidores de Caspase , Morte Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Glioma , Humanos , Proteínas Inibidoras de Apoptose , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , SurvivinaRESUMO
Anticancer activity of silibinin, a flavonoid, has been demonstrated in various cancer cell types. However, the underlying mechanism and in vivo efficacy in glioma were not elucidated. The present study was undertaken to determine the effect of silibinin on glioma cell proliferation in vitro and to examine whether silibinin inhibits tumor growth in vivo. Silibinin resulted in inhibition of proliferation in a dose- and time-dependent manner, which was largely attributed to cell death. Silibinin induced a transient increase in intracellular Ca2+ followed by an increase in reactive oxygen species (ROS) generation. The silibinin-induced cell death was prevented by EGTA, calpain inhibitor and antioxidants (N-acetylcysteine and Trolox). Western blot analysis showed that silibinin also induced ROS-dependent activation of extracellular signal-regulated kinase, p38 kinase, and c-Jun N-terminal kinase. Inhibitors of these kinases prevented the silibinin-induced cell death. Silibinin caused caspase activation and the silibinin-induced cell death was prevented by caspase inhibitors. Glioma cell migration was also decreased by silibinin treatment. Oral administration of silibinin in animals with subcutaneous U87MG glioma cells reduced tumor volume. Subsequent tumor tissue analysis showed a decrease in Ki-67 positive cells, an increase in TUNEL-positive cells, and caspase activation. These results indicate that silibinin induces a caspase-dependent cell death via Ca2+/ROS/MAPK-mediated pathway in vitro and inhibits glioma growth in vivo. These data suggest that silibinin may serve as a potential therapeutic agent for malignant human gliomas.
Assuntos
Antioxidantes/farmacologia , Neoplasias Encefálicas/patologia , Sinalização do Cálcio/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Glioma/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/efeitos dos fármacos , Western Blotting , Cálcio/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Indicadores e Reagentes , Silibina , Silimarina/farmacologiaRESUMO
We have previously reported the cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) induces renal proximal epithelial cell death through NF-kappaB inhibition. However, the upstream and down-stream signaling pathways that NF-kappaB inhibition mediates 15d-PGJ(2)-induced apoptosis remain to be defined. In the present study, we determined whether NF-kappaB inhibition induces cell death through the mitochondrial apoptotic pathway and whether protein kinase A (PKA) functions upstream of NF-kappaB inhibition by 15d-PGJ(2). The role of NF-kappaB inhibition in this apoptotic pathway was evaluated using NF-kappaB p65 transfected cells. 15d-PGJ(2) induced cell death by a PPARgamma-independent mechanism and the cell death was prevented by NF-kappaB p65 transfection. 15d-PGJ(2) treatment caused disruption of mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, suggesting that 15d-PGJ(2) induces cell death through a mitochondria-dependent apoptotic mechanism. These changes by 15d-PGJ(2) were attenuated by NF-kappaB p65 transfection. 15d-PGJ(2) treatment resulted in an increase in Bax expression, which were blocked by NF-kappaB p65 transfection. 15d-PGJ(2) treatment caused PKA inhibition and 15d-PGJ(2)-induced cell death was enhanced by the PKA specific inhibitor H89. Inhibition of NF-kappaB by 15d-PGJ(2) was prevented by addition of forskolin, a PKA activator. Taken together, these results suggest that PKA-dependent NF-kappaB inhibition stimulates 15d-PGJ(2)-mediated mitochondrial apoptotic pathway through alterations in expression of the NF-kappaB target genes Bax.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Células Epiteliais/efeitos dos fármacos , Túbulos Renais Proximais/patologia , Mitocôndrias/fisiologia , NF-kappa B/antagonistas & inibidores , Prostaglandina D2/análogos & derivados , Animais , Western Blotting , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Genes Reporter , Imuno-Histoquímica , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/enzimologia , Túbulos Renais Proximais/metabolismo , Luciferases/genética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/metabolismo , NF-kappa B/genética , Gambás , Prostaglandina D2/farmacologia , Prostaglandina D2/fisiologiaRESUMO
We have previously demonstrated that the synthetic peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist ciglitazone induces apoptosis accompanied by activation of p38 mitogen-activated protein kinase (MAPK) and nuclear translocation of apoptosis inducing factor (AIF) in opossum kidney (OK) renal epithelial cells. However, the precise mechanism by which ciglitazone induces activation of p38 MAPK and the role of AIF in the induction of the apoptosis are not defined. This study was therefore undertaken to determine whether the roles of reactive oxygen species (ROS) generation and intracellular Ca(2+) in the ciglitazone-induced activation of p38 MAPK and whether AIF nuclear translocation is responsible for the ciglitazone-induced apoptosis in OK renal epithelial cells. Ciglitazone caused generation of ROS and an increase in intracellular Ca(2+). Ciglitazone-induced cell death was reduced by the antioxidant Trolox, the Ca(2+) chelator EGTA, and the store-operated Ca(2+) channels (SOCC) blocker lanthanum chloride (La(3+)), indicating involvement of ROS and Ca(2+) in the ciglitazone-induced cell death. Ciglitazone-induced intracellular Ca(2+) increase was decreased by Trolox, while ROS generation was not affected by EGTA and La(3+), suggesting that ROS generation promote the increase of intracellular Ca(2+). Transfection of small interfering RNA (siRNA) of p38 MAPK or vector expressing microRNA (miRNA) of AIF prevented the ciglitazone-induced cell death. Activation of p38 MAPK, mitochondrial membrane depolarization, and AIF nuclear translocation induced by ciglitazone were inhibited by Trolox, EGTA and La(3+). Taken together, these results suggest that ROS-dependent intracellular Ca(2+) increase is responsible for activation of p38 MAPK and nuclear translocation of AIF by ciglitazone.
Assuntos
Fator de Indução de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Hipoglicemiantes/toxicidade , Rim/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tiazolidinedionas/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Antioxidantes/farmacologia , Fator de Indução de Apoptose/genética , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Quelantes/farmacologia , Cromanos/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Rim/enzimologia , Rim/patologia , Lantânio/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Gambás , PPAR gama/agonistas , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
PURPOSE:: Development of a new, fiber-free, single-use endo-illuminator for pars plana vitrectomy as a replacement for fiber-based systems with external light sources. The hand-guided intraocularly placed white micro light-emitting diode is evaluated for its illumination properties and potential photochemical and thermal hazards. METHODS:: A micro light-emitting diode was used to develop a single-use intraocular illumination system. The light-source-on-tip device was implemented in a prototype with 23G trocar compatible outer diameter of 0.6 mm. The experimental testing was performed on porcine eyes. All calculations of possible photochemical and thermal hazards during the application of the intraocular micro light-emitting diode were calculated according to DIN EN ISO 15007-2: 2014. RESULTS:: The endo-illuminator generated a homogeneous and bright illumination of the intraocular space. The color impression was physiologic and natural. Contrary to initial apprehension, the possible risk caused by inserting a light-emitting diode into the intraocular vitreous was much smaller when compared to conventional fiber-based illumination systems. The photochemical and thermal hazards allowed a continuous exposure time to the retina of at least 4.7 h. CONCLUSION:: This first intraocular light source showed that a light-emitting diode can be introduced into the eye. The system can be built as single-use illumination system. This light-source-on-tip light-emitting diode-endo-illumination combines a chandelier wide-angle illumination with an adjustable endo-illuminator.
Assuntos
Luz , Iluminação/instrumentação , Semicondutores , Vitrectomia , Animais , Desenho de Equipamento , Miniaturização , Suínos , Cirurgia VitreorretinianaRESUMO
Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been reported to induce apoptosis in a variety of cell types including renal proximal epithelial cells. However, the underlying mechanism of cell death induced by PPARgamma agonists has not been clearly defined in renal proximal tubular cells. This study was therefore undertaken to determine the mechanism by which ciglitazone, a synthetic PPARgamma agonist, induces apoptosis in opossum kidney (OK) cells, an established renal epithelial cell line. Ciglitazone treatment induced apoptotic cell death in a dose- and time-dependent manner. Ciglitazone caused a transient activation of ERK and sustained activation of p38 MAP kinase. Ciglitazone-mediated cell death was attenuated by the p38 inhibitor SB203580 and transfection of dominant-negative form of p38, but not by the MEK inhibitor U0126, indicating that p38 MAP kinase activation is involved in the ciglitazone-induced cell death. Although ciglitazone-induced caspase-3 activation, the ciglitazone-mediated cell death was not affected by the caspase-3 inhibitor DEVD-CHO. Ciglitazone-induced mitochondrial membrane depolarization and apoptosis-inducing factor (AIF) nuclear translocation and these effects were prevented by the p38 inhibitor. These results suggest that ciglitazone induces caspase-independent apoptosis through p38 MAP kinase-dependent AIF nuclear translocation in OK renal epithelial cells.
Assuntos
Fator de Indução de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Tiazolidinedionas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cromanos/farmacologia , Ácido Clofíbrico/análogos & derivados , Ácido Clofíbrico/farmacologia , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Ácidos Fíbricos , Citometria de Fluxo , Imunofluorescência , Fase G1/efeitos dos fármacos , Imidazóis/farmacologia , Túbulos Renais/citologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Gambás , PPAR gama/agonistas , Piridinas/farmacologia , TroglitazonaRESUMO
The cyclopentenone prostaglandin 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) induces apoptosis in various cell types. However, the underlying mechanism of 15d-PGJ2-induced apoptosis is not fully understood. The present study was undertaken to determine the molecular mechanism by which 15d-PGJ2 induces apoptosis in MC3T3-E1 mouse osteoblastic cells. 15d-PGJ2 caused a concentration- and time-dependent apoptotic cell death. 15d-PGJ2 induced a transient activation of ERK1/2 and sustained activation of JNK. 15d-PGJ2-induced cell death was prevented by the JNK inhibitor SP6001, but not by inhibitors of ERK1/2 and p38. JNK activation by 15d-PGJ2 was blocked by antioxidants N-acetylcysteine (NAC) and GSH. 15d-PGJ2 caused ROS generation and 15d-PGJ2-induced cell death was prevented by antioxidants, suggesting involvement of ROS generation in 15d-PGJ2-induced cell death. 15d-PGJ2 triggered the mitochondrial apoptotic pathway indicated by enhanced Bax expression, loss of mitochondrial membrane potential, cytochrome c release, and caspase-3 activation. The JNK inhibitor blocked these events induced by 15d-PGJ2. Taken together, these results suggest that the 15d-PGJ2 induces cell death through the mitochondrial apoptotic pathway dependent of ROS and JNK activation in osteoblastic cells.
Assuntos
Apoptose/efeitos dos fármacos , Fatores Imunológicos/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mitocôndrias/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Prostaglandina D2/análogos & derivados , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , PPAR gama/metabolismo , Prostaglandina D2/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Troglitazone, a PPARgamma agonist, has been reported to induce cell death on different cell types. However, its mechanism of action remains unclear. The present study was undertaken to investigate the effect of troglitazone on cell death and to determine its underlying mechanism in MC3T3-E1 cells, an established osteoblast cell line. Troglitazone induced loss of cell viability in a dose- and time-dependent manner, which was accompanied by apoptosis. Troglitazone increased reactive oxygen species (ROS), but troglitazone-induced cell death was not affected by the antioxidant N-acetylcysteine, suggesting that the ROS generation is not involved in the cytotoxicity of troglitazone. Troglitazone-induced cell death was prevented by the PPARgamma antagonist GW9662. Troglitazone treatment inhibited activation of extracellular signal-regulated protein kinase (ERK) and stimulated p38 activation. Troglitazone-induced cell death was increased by the ERK inhibitor U0126 and prevented by transfection with constitutively active MEK1 and the p38 inhibitor SB203580. Troglitazone induced depolarization of mitochondrial membrane potential and its effect was blocked by SB203580 and GW9662. Caspase-3 was activated by troglitazone treatment and pharmacological inhibition of caspase blocked troglitazone-induced cell death. Taken together, these data suggest that troglitazone induces apoptosis via a caspase-dependent mechanism associated with down-regulation of ERK and up-regulation of p38.
Assuntos
Apoptose/efeitos dos fármacos , Cromanos/toxicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Osteoblastos/efeitos dos fármacos , Tiazolidinedionas/toxicidade , Células 3T3 , Anilidas/farmacologia , Animais , Western Blotting , Butadienos/farmacologia , Caspase 3/metabolismo , Inibidores de Caspase , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Hipoglicemiantes/toxicidade , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Nitrilas/farmacologia , Osteoblastos/citologia , Osteoblastos/metabolismo , PPAR gama/agonistas , PPAR gama/antagonistas & inibidores , PPAR gama/metabolismo , Piridinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , TroglitazonaRESUMO
The extracellular signal-regulated kinase (ERK) and Akt have been reported to be activated by ischemia/reperfusion in vivo. However, the signaling pathways involved in activation of these kinases and their potential roles were not fully understood in the postischemic kidney. In the present study, we observed that these kinases are activated by hypoxia/reoxygenation (H/R), an in vitro model of ischemia/reperfusion, in opossum kidney (OK) cells and elucidated the signaling pathways of these kinases. ERK and Akt were transiently activated during the early phase of reoxygenation following 4-12h of hypoxia. The ERK activation was inhibited by U0126, a specific inhibitor of ERK upstream MAPK/ERK kinase (MEK), but not by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), whereas Akt activation was blocked by LY294002, but not by U0126. Inhibitors of epidermal growth factor receptor (EGFR) (AG 1478), Ras and Raf, as well as antioxidants inhibited activation of ERK and Akt, while the Src inhibitor PP2 had no effect. PI3K/Akt activation was shown to be associated with up-regulation of X chromosome-linked inhibitor of apoptosis (XIAP), but not survivin. Reoxygenation following 4-h hypoxia-stimulated cell proliferation, which was dependent on ERK and Akt activation and was also inhibited by antioxidants and AG 1478. Taken together, these results suggest that H/R induces activation of MEK/ERK and PI3K/Akt/XIAP survival signaling pathways through the reactive oxygen species-dependent EGFR/Ras/Raf cascade. Activation of these kinases may be involved in the repair process during ischemia/reperfusion.
Assuntos
Células Epiteliais/enzimologia , Rim/enzimologia , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Butadienos/farmacologia , Hipóxia Celular , Proliferação de Células , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Rim/citologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Nitrilas/farmacologia , Gambás , Proteínas Proto-Oncogênicas c-raf/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/enzimologia , Transdução de Sinais , Transfecção , Proteínas ras/metabolismoRESUMO
Sphingosylphosphorylcholine (SPC) is a bioactive lipid molecule involved in a variety of cellular responses. In the present study, we demonstrated that treatment of human adipose tissue-derived mesenchymal stem cells (hATSCs) with D-erythro-SPC resulted in apoptosis-like cell death, as demonstrated by decreased cell viability, DNA strand breaks, the increase of sub-G1 fraction, cytochrome c release into cytosol, and activation of caspase-3. In contrast, the exposure of hATSCs to L-threo-SPC did not induce the cell death, suggesting that the SPC-induced cell death was selective for the D-erythro-stereoisomer of SPC. The D-erythro-SPC-induced cell death was prevented by DEVD-CHO, a caspase-3 specific inhibitor, and Z-VAD-FMK, a general caspase inhibitor, suggesting that the SPC-induced cell death of hATSCs occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, D-erythro-SPC treatment stimulated the activation of mitogen-activated protein kinases, such as ERK and c-Jun NH2-terminal protein kinase (JNK), and the D-erythro-SPC-induced cell death was completely prevented by pretreatment with the MEK inhibitor, U0126, but not by pretreatment with the JNK inhibitor, SP600125, and the p38 MAPK inhibitor, SB202190, suggesting a specific involvement of ERK in the D-erythro-SPC-induced cell death. Pretreatment with U0126 attenuated the D-erythro-SPC-induced release of cytochrome c. From these results, we suggest that ERK is involved in the SPC-induced cell death of hATSC through stimulation of the cytochrome c/caspase-3-dependent pathway.